Rabies viral encephalitis is highly lethal with only a few documented survivors of the disease. While anti-rabies reagents are available that are effective either prior to, or in the first days following exposure, they require multiple doses and are ineffective once the virus has reached the CNS. We have recently developed a live- attenuated recombinant rabies virus (RV) vaccine, 3-GAS, that has a unique safety profile and can prevent a lethal infection with wild-type RV in mice when used at a single dose for either pre- or post-exposure immunization. The primary objectives of this project are to establish an optimal treatment regimen whereby 3- GAS administration triggers immune clearance of wild-type RV from the CNS and identify biomarkers associated with the various elements of protective immunity that are predictive of successful treatment. In addition, we will elucidate the mechanisms that are responsible for the 3-GAS vaccine-mediated clearance of pathogenic RV from the CNS. This knowledge will enable us to further enhance the efficacy of 3-GAS by constructing variants better target RV-clearing immune effectors to the CNS. Modifications will include the expression of cytokines that alter blood-brain barrier function and autophagy as well as modify virus tissue specificity. The secondary objectives of this project are to determine if 3-GAS and its derivatives are safe, sufficiently stable and immunogenic so that they can be used for pre- and post-exposure prophylaxis of human rabies. Particularly important here is to establish whether 3-GAS and 3-GAS variants are effective with a single dose and have therapeutic activity when clinical rabies is developing and conventional post-exposure prophylaxis with inactivated vaccines is no longer effective.